Oral insulin composition and methods of making and using thereof

ABSTRACT

A method of lowering blood glucose in a mammal includes orally administering a therapeutically effective amount of crystallized dextran microparticles and insulin to the mammal to lower blood glucose of the mammal. The composition may be a one phase or a structured multi-phase composition for controlled release of insulin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/792,376, filed Mar. 4, 2004, which claims priority to U.S.Provisional Patent Application Ser. No. 60/451,245, filed Mar. 4, 2003;U.S. Provisional Patent Application Ser. No. 60/467,601, filed May 5,2003; U.S. Provisional Patent Application Ser. No. 60/469,017, filed May9, 2003; and U.S. Provisional Patent Application Ser. No. 60/495,097,filed Aug. 15, 2003, the disclosures of each of the foregoingapplications being incorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to insulin compositions andspecifically to an oral insulin composition containing insulin andcrystallized dextran microparticles.

BACKGROUND OF THE INVENTION

Dextrans are high molecular weight polysaccharides synthesized by somemicro organisms or by biochemical methods. Dextran with averagemolecular weight of about 75 kDa has a colloid osmotic pressure similarto blood plasma, so its aqueous solutions are used clinically as plasmaexpanders. Cross-linked dextrans in the form of beads are the basis for“Sephadex”® that is used in the GPC of proteins and for “Cytodex”®developed by Pharmacia to fulfill the special requirements of amicro-carrier cell culture. For example, U.S. Pat. Nos. 6,395,302 and6,303,148 (Hennink et al.) disclose attaching various biomaterials tocross-linked dextran particles. However, beads based on cross-linkeddextran generally cannot be used for implant manufacturing owing totheir potential toxicity due to the application of cross-linking agents(Blain J. F., Maghni K., Pelletier S. and Sirois P. Inflamm. Res. 48(1999): 386-392).

U.S. Pat. No. 4,713,249 (Schroder) describes a method of producing adepot matrix for biologically active substances. According to thispatent, the depot matrix allegedly consists of carbohydratemicroparticles, stabilized by crystallization, which implies usingnon-covalent bonds. The following process for producing the allegedcrystallized carbohydrate microparticles is described by Schroder. Asolution of a polymeric carbohydrate and a biologically-active substanceis formed in one or more hydrophilic solvents. Then the mixture of thecarbohydrate and the biologically active substance is emulsified in aliquid hydrophobic medium to form spherical droplets. The emulsion isthen introduced into a crystallizing medium comprising acetone, ethanolor methanol to form spheres having a non-covalently cross-linkedcrystalline polymeric carbohydrate matrix, said matrix incorporating0.001-50% by weight of the biologically-active substance. Thus, thebiologically active substance is provided into the solution prior tocrystallizing the spheres. Schroder does not describe the microstructureof the microparticles made by the multi-step method. Schroder'smulti-step method is complex and uses organic solvents that arepotentially toxic to cells and need to be removed. Furthermore,Schroder's composition is designed for injection, which is aninconvenient and painful method of administering the composition.

BRIEF SUMMARY OF THE INVENTION

A method of lowering blood glucose in a mammal includes orallyadministering a therapeutically effective amount of crystallized dextranmicroparticles and insulin to the mammal to lower blood glucose of themammal. The composition may be a one phase or a structured multi-phasecomposition for controlled release of insulin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a photograph of crystallized dextran microparticlesspontaneously formed in 55.0% (W/W) aqueous solution of dextran with MW70.0 kDa.

FIG. 2A is a photograph of a cross-section of crystallized dextranmicroparticles shown in FIG. 1.

FIG. 2B is a photograph of a cross-section of a microparticle shown inFIG. 2A, Microporous structure of the microparticle can be seen.

FIG. 3 is a photograph of aggregates of crystallized dextranmicroparticles.

FIG. 4 is a photograph of a slow release of the fluorescently labeledmacromolecules from the implant which includes crystallized dextranmicroparticles into mouse muscle tissue on the 14.sup.th day afterintermuscular injection.

FIG. 5 is a photograph of an emulsion of aqueous solution of PEG inaqueous solution of dextran (MW 500 kDa) containing crystallized dextranmicroparticles shown in FIG. 1.

FIG. 6 is a photograph of an emulsion of aqueous solution of dextran (MW500 kDa) containing crystallized dextran microparticles shown in FIG. 1in aqueous solution of PEG.

FIG. 7 is a photograph of an intramuscular injection of emulsion ofaqueous solution of PEG in aqueous solution of dextran (MW 500 kDa)containing crystallized dextran microparticles shown in FIG. 1.

FIG. 8 is a photograph of a subcutaneous injection of emulsion ofaqueous solution of PEG in aqueous solution of dextran (MW 500 kDa)containing crystallized dextran microparticles shown in FIG. 1.

FIGS. 9A and 9C schematically illustrate partition behavior of differenttypes of particles and phases in an aqueous two phase system.

FIG. 9B is a photograph of a cross section of an implant structure basedon the two phase system.

FIG. 10 schematically illustrates therapeutic agent delivery methodsaccording to an embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present inventor discovered that a composition of porous (i.e.,microporous) microparticles, such as crystallized dextranmicroparticles, may be used as a vehicle for oral delivery of insulin.The composition may be a one phase composition or a multi-phasecomposition which forms a structured implant in a mammal.

The first section below describes the preferred crystallized dextranmicroparticles, the second section describes formation of the structuredimplant from a multi-phase composition, and the following sectionsdescribe specific examples of oral administration of the compositioninto mammals and methods of making the oral insulin composition.

A. Crystallized Dextran Microparticles

The present inventor has experimentally found that crystallized dextranmicroparticles with an average diameter ranging from 0.5 to 3.5 micronswere spontaneously formed in concentrated aqueous solutions of dextrans(40-65% W/W) with molecular weights ranging from 1.0 to 200.0 kDa, attemperature ranging from 20-90° C. If it is desired to form themicroparticles at room temperature, then 2 to 18 kDa dextran solutionsmay be used. Of course, the microparticles may also be formed from 2 to18 kDa solutions at temperatures above room temperature, if desired. Themicroparticles may be spontaneously formed from higher molecular weightdextran solutions, such as 20 to 75 kDa solutions, at highertemperatures above room temperature, such as about 40 to about 70° C.The microparticles may have any suitable shape such as a regular or anirregular shape, but are preferably spherical in shape, and arepreferably 10 microns in diameter or less, such as 0.5 to 5 microns.

Transmission Electron Microscopy revealed the microporous structure ofthe crystallized dextran microparticles (see FIGS. 2A, 2B). Preferably,the microparticle porosity is at least 10 percent by volume, such asabout 10 to about 50 percent, more preferably about 20 to about 40percent. Thus, the structure comprises microporous microparticles withareas of macroporosity located between the particles.

Spray drying of aqueous suspensions of the crystallized dextranmicroparticles has shown the possibility to produce substantiallyspherical aggregates of crystallized dextran microparticles with adiameter ranging from 10.0 to 150.0 microns (see FIG. 3).

A non limiting example of a method of forming the dextran microparticlesis as follows. 50.0 g of dextran T40 (40 kDa molecular weight) fromAmersham Biosciences is added to 50.0 g of sterile distilled water in a500 ml lab beaker to obtain 50% w/w solution under laminar flow. Themixture is stirred at 60° C. (water bath) on a magnetic stirrer at 50rpm until the dextran is completely dissolved and a clear solution isobtained. The solution can be vacuumed to remove all air inclusions. Theclear solution is placed in lab oven at 60° C. under a Tyvek® lid. 3.5hours later, a turbid viscous suspension is developed as a result offormation of crystallized dextran microparticles.

To eliminate non-crystallized dextran, the microparticles are washed bycentrifugation, for example 3,000 g, 30 min, with 3×250 ml of distilledsterile water, or by filtration of diluted suspension of microparticles,for example one part microparticles and 10 parts water (3×250 ml ofdistilled sterile water through sterilization filter). Thecentrifugation/washing is done under laminar flow. The microparticlesare placed in 500 ml lab beaker under a Tyvek® lid and dried at 60° C.in lab oven for 8 hours to reach a moisture level of about 5%. Theresulting dry powder consists of particles with a mean diameter of about2 microns.

The slow release of macromolecules from implants has been demonstratedin experiments where macromolecules were dissolved in aqueoussuspensions of crystallized dextran microparticles or their aggregatesbefore injections. FIG. 4 shows an implant containing fluorescentlylabeled macromolecules (FITC-dextran, MW 500 kDa) and slow release ofthe macromolecules from the implant into a mouse muscle tissue on the14.sup.th day after the intermuscular injection.

B. Two-Phase System

Self assembled structures based on crystallized dextran microparticlesand their aggregates may be formed based on two phase systems.

For example, in the case of oil, a special kind of structure can beformed where the oil core is surrounded with a shell composed ofcrystallized dextran microparticles or aggregates thereof dispersed inwater or aqueous solutions of organic polymers such as polysaccharides(e.g. dextrans). The structure described can be designated as a capsule.It should be noted that the shell may comprise a roughly sphericalshaped shell which results when the capsule is surrounded by tissue.However, when the capsule is located near a barrier, such as asubstrate, bone or intestine wall, the capsule may comprise a corelocated between one or more walls of microparticles on one side and thebarrier on the other side. Furthermore, while oil is used as anillustrative example, the core may comprise other materials, such asother polymers, cells, etc.

To form the capsule structure, two-phase aqueous systems are applied.When aqueous solutions of different polymers are mixed above certainconcentrations they frequently form immiscible-liquid two-phasesolutions. Each of the phases usually consists of more than 90% waterand can be buffered and made isotonic. If a cell or particle suspensionis added to such a system, the cells or particles are frequently foundto have partitioned unequally between phases. This preferentialpartition behavior can be used as a basis for separation procedures fordiffering cell populations or particles since partition in these systemsis determined directly by cell or particle surface properties. Cells orparticles which do not have identical surface properties exhibitsufficiently different partition behavior.

The competitive adsorption of the two polymer phase depends on thechemical nature of the polymers. A two-phase polymer method has beenapplied to separate or partition cells, proteins, nucleic acids andminerals (“Partitioning in Aqueous Two-Phase Systems”, 1985, eds., H.Walter, D. Brooks, and D. Fisher, pubis. Academic Press).

The experiments with the distribution of crystallized dextranmicroparticles in phase systems derived from, for instance,dextran/polyethylene glycol (PEG) mixtures, revealed that the dextranmicroparticles prefer to be in the dextran phase, while another PEGphase can be dispersed in this dextran phase to form a W/W emulsion andvice versa in the case when the volume of the PEG phase is bigger thanthe volume of the dextran phase, as shown in FIGS. 5 and 6.

FIG. 5 is a photograph of an emulsion of aqueous solution of PEG inaqueous solution of dextran containing crystallized dextranmicroparticles. In the structure of FIG. 5, the volume of the PEG phaseis less than the volume of the dextran phase. The dextran phase containsthe dextran and the crystallized dextran microparticles. Thus, the PEGphase forms into one or more sphere shaped cores surrounded by dextrandextran microparticle shells (i.e., a closed pore structure).

FIG. 6 is a photograph of an emulsion of aqueous solution of dextrancontaining crystallized dextran microparticles in aqueous solution ofPEG, where the volume of the PEG phase is greater than the volume of thedextran phase. In this case, the dextran phase forms into one or moresphere shaped cores containing the dextran microparticles surrounded bya PEG phase (i.e., an open pore structure that is forming in vivo whilePEG dissipates in tissue liquid). As can be seen in FIG. 6, the smallervolume (droplet) dextran phase forms into a large sphericaldextran/dextran microparticle core (bottom right of FIG. 6) to whichsmaller spheres comprising dextran/dextran microparticles are joiningand fuse with.

Thus, when the ratio of the volume of the first phase (such as the PEGphase and its inclusions, such as a therapeutic agent) to the volume ofthe second phase (such as the dextran phase and its inclusions, such asthe dextran microparticles) is less than one, then the capsule forms byself assembly with a first phase core surrounded by a second phaseshell. If the composition contains a therapeutic agent, such as insulin,which prefers to partition into the PEG phase, and the dextranmicroparticles which prefer to partition into the dextran phase, thenthe therapeutic agent selectively partitions into the PEG core while themicroparticles selectively partition into and form the shell around thePEG core by self assembly.

The emulsion can be prepared by the mixing of separately prepareddextran and PEG phases and both can be suspensions of different types ofparticles that prefer to be in the PEG phase or in the dextran phaserespectively. The principle is that the partition of particles intodifferent polymer phases depends on their surface structure andinterfacial energy of the particles in the polymer solutions.

Injection of aqueous two phase systems containing crystallized dextranmicroparticles into tissues of experimental animals revealed theformation of implants with the capsule structure as shown in FIGS. 7 and8. The volume of the dextran phase is greater than the volume of the PEGphase in the two-phase system. Both FIGS. 7 and 8 show that a capsulewith a PEG core and a dextran/dextran microparticle shell forms by selfassembly in vivo (i.e., after injection into mammal tissue). The shellcomprises macroporous regions between adjacent microparticles as well asmicroporous regions in the microparticles themselves.

A non limiting example of a method of forming a capsule structure from atwo phase system is as follows. 10 g of dextran T40 (40 kDa molecularweight) and 2 g of PEG are dissolved in 88 ml of (Actrapid®) insulinsolution containing 1,000 UI to which 25 g of crystallized dextranmicroparticles are added. These steps are performed under laminar flowconditions. The mixture is stirred on a magnetic stirrer at 100 rpm atroom temperature for 30 minutes to form a homogeneous mixture (i.e., asuspension). 1.0 g of the suspension contains 8 UI of insulin.

It should be noted that the dextran microparticles may be prepared froma different molecular weight dextran solution than the dextran solutionwhich is provided into the two phase system. Thus, the crystallizeddextran microparticles may be formed in a lower molecular weight dextransolution, such as a 2 to 20 kDa solution, than the dextran solutionwhich is provided in the two phase system, which may be a 40 to 500 kDadextran solution, such as a 40 to 75 kDa solution. This is advantageousbecause the higher molecular weight dextran solutions, such as 40 and 70kDa solutions, have received wider regulatory approval and can be usedto form a shell of a capsule at lower concentrations. The lowermolecular weight solutions may be used to decrease the crystallizationtime without the lower molecular weight dextran solution actually beingprovided in vivo. Furthermore, lower molecular weight microparticles maydissolve easier in vivo.

The capsule structure formed from a two phase system is advantageousbecause it allows for a more even and prolonged release of thetherapeutic agent from the core than from a composition comprising asingle phase containing the microparticles. Furthermore, it is believedthat by using the capsule structure, a lower amount of microparticlesmay be needed to achieve the same or better timed release of atherapeutic agent than if a single phase system is used. Furthermore, bycontrolling the amount of microparticles in the two phase system, it isbelieved that the thickness of the microparticle shell may becontrolled. A thicker shell results from a larger amount ofmicroparticles in the two phase system. Thus, the amount, durationand/or timing of the release of the therapeutic agent from the capsulecore may be controlled by controlling the thickness of the shell.Therefore, the release profile of the therapeutic agent may becustomized for each patient or groups of patients.

It should be noted that while PEG and dextran are used as examples ofthe materials of the two phases, any other suitable materials which showthe following partition behavior may be used instead. FIG. 9Aschematically illustrates partition behavior of different types ofparticles in an aqueous two phase system. For example, three types ofmolecules or molecular aggregates, which are preferably particles 10, 12and 14, and two phases 16 and 18 are shown in FIG. 9A. However, theremay be two or more than three types of particles. The particles may bemicroparticles such as microspheres or nanospheres prepared from organicand/or inorganic materials, liposomes, living cells, viruses andmacromolecules. The first type particles 10 preferentially segregateinto the first phase 16. The second type particles 12 preferentiallysegregate to the boundary of the first 16 and second 18 phases. Thethird type particles 14 preferentially segregate into the second phase18. Thus, by analogy to the previous non-limiting example, the firstparticles 10 may comprise a therapeutic agent, the second 12 and/or thethird 14 particles may comprise crystallized dextran microparticles, thefirst phase 16 may comprise a PEG phase and the second phase 18 maycomprise a dextran phase.

If a smaller amount of the first phase 16 is provided into a largeramount of the second phase 18, as shown in area 20 of FIG. 9A, then acapsule type structure forms comprising discreet spheres of the firstphase 16 containing a concentration of the first type particles 10,located in a second phase 18. The second type particles 12 may belocated at the interface of the phases 16, 18 and act as a shell of thecapsule. Particles 14 are dispersed in the second phase 18 and/or form ashell of the capsule.

In contrast, if a smaller amount of the second phase 18 is provided intoa larger amount of the first phase 16, as shown in area 22 of FIG. 9A,then a capsule type structure forms comprising discreet spheres of thesecond phase 18 containing a concentration of the third type particles14, located in a first phase 16. The second type particles 12 may belocated at the interface of the phases 16, 18 and act as a shell of thecapsule. Particles 10 are dispersed in the first phase 16 and/or form ashell of the capsule. The two phase systems 20 and 22 may be used as animplant, such as by being delivered into a mammal, such as an animal orhuman. Thus, the capsule forms a structured, three dimensional implant,with the core acting as a reservoir or depot for controlled release ofthe therapeutic agent through the shell. In contrast, an implant with aneven distribution of microparticles is an unstructured implant. Itshould be noted that the structure formed for orally delivered two phasesystems may be generally described as a structured suspension comprisinga dispersed PEG phase and a continuous dextran phase.

Furthermore, particles 10, 12 and 14 may be substituted by a liquidmaterial (e.g. oils) or macromolecules which selectively partition intoone of the phases. For example, a therapeutic agent, such as insulin,may be partitioned in PEG phase of the PEG/dextran two phase system.Since insulin selectively partitions into the PEG phase, the PEG phaseforms an insulin containing core of a capsule structure. It should benoted that while certain particles and therapeutic agents selectivelypartition, the term “selectively partitioned” does not necessarily meanthat 100 percent of the particles or therapeutic agent partition intoone of the phases. However, a majority of the selectively partitionedspecie, preferably 80% of the partitioned specie, partitions into one ofthe phases. For example, while a majority of insulin partitions into thePEG phase, a portion of insulin may remain in the dextran phase.

FIG. 9B illustrates a scanning electron microscope image of a crosssection of an implant structure based on the two phase systemschematically illustrated in FIG. 9A. A two phase aqueous compositioncomprising a first dextran phase, a second PEG phase and crystallizeddextran microparticles was injected into sepharose gel. This gel'scomposition mimics mammal tissue by stopping crystallized dextranmicroparticles diffusion from the injection side. The image in FIG. 9Billustrates the formation of a core-shell implant structure. The corecomprises regions 30 and 32 surrounded by a shell 34. Region 30 is avoid that is filled with a PEG phase region prior to cutting the gel forcross sectional SEM imaging. The PEG phase region drips out of the gelwhen the gel is cut during cross sectioning. Region 32 is an outerportion of the core comprising PEG droplets located in the crystallizeddextran microparticles. Region 34 is the shell comprising thecrystallized dextran microparticles which surrounds and holds in placethe PEG containing core.

Without wishing to be bound by a particular theory, the present inventorbelieves that the core-shell structure shown in FIG. 9B forms by selfassembly as shown schematically in FIG. 9C. While the first 16 andsecond 18 phases, such as aqueous solutions of different, incompatiblepolymers, are in a suitable storage container 19, such as in a glassbeaker or vial, one phase 16 rises above the other phase 18. When thetwo phase composition is injected into a material which restricts freeflow of the phases 16 and 18, such as mammal tissue or a substratematerial, such as a gel which mimics the tissue, the composition selfassembles into the core-shell structure. First, the phase that ispresent in the smaller volume forms into approximate spherical shapes,as shown in the middle portion of FIG. 9C. Then the spherical shapesjoin to form approximately spherical cores of one phase surrounded byshells of the other phase, as shown in the bottom of FIG. 9C. While atwo phase system example of a multiphase system has been illustrated,the multiphase system may have more than two phases if desired.

C. Oral Insulin Delivery Vehicle

The present inventor discovered that a composition of porous (i.e.,microporous) microparticles may be used as a vehicle for oral deliveryof insulin. The porous microparticles may be any suitable porousmicroparticles which enable oral administration of insulin with asignificant reduction in blood glucose, such as at least a 5% reduction,for example at least a 30% reduction within 60 minutes of oraladministration. Preferably, the microparticles are bioadhesiveparticles, such as particles which adhere at least temporarily to mammalintestine walls, to allow insulin deliver through the intestine wall.Most preferably, the porous microparticles comprise the crystallizeddextran microparticles described above.

In one preferred embodiment of the present invention shown in FIG. 10,the present inventor has discovered that an aqueous suspension ofcrystallized dextran microparticles 12, 14 and insulin 46 orallyadministered to mammals 53, such as rabbits, was about equally aseffective in reducing blood glucose levels as an intramuscular injectionof insulin alone. FIG. 10 schematically illustrates the insulin 46permeating through mammal 53 intestine 52 walls from the orallyadministered composition 54 comprising the microparticles. Since rabbitsare a common model for humans in drug testing, the present inventorbelieves that a liquid or solid composition 54 comprising crystallizeddextran microparticles and insulin, such as an aqueous suspension, asolution, a tablet or a capsule, would also be effective in reducingblood glucose levels in human beings when orally administered.

The following examples illustrate oral insulin delivery usingcrystallized dextran microparticles. The study involved Chinchillarabbits (2.3.+−.0.2 kg) and the observation of their response to orallyadministered aqueous suspensions consisting of crystallized dextranmicroparticles prepared according to the method described herein andhuman recombinant insulin.

3.0 g of Dextran T20 (Pharmacia, Uppsala, Sweden) was dissolved in 2.0 gof water and placed in box at temperature 60° C. Three hours later,crystallized dextran microparticles were washed by centrifugation at3,000 g with 3×5.0 ml of water. Then the crystallized dextranmicroparticles were suspended in 2.0 ml of water and allowed to dry atroom temperature. The resulting dry powder was used to prepare aninsulin containing suspension for the oral insulin delivery experiment.Insulin containing suspensions were prepared by the mixing of 250 mg ofthe microparticles; 0.3 ml (12 UI) or 0.6 ml (24 UI) of insulin(NovoNordisk Actrapid HM Penfill, 40 UI/ml); and distilled water toreach a volume of 2.0 ml.

Samples of the suspension (2.0 ml) were introduced into the rabbits'throats with a catheter followed by the introduction of 10.0 ml ofdrinking water. Animals were not fed for 3 hours before the suspension'sintroduction. Samples of blood were taken from the rabbit's ear vein andanalyzed for glucose concentrations. Blood glucose was measured usingthe glucose oxidase method on a “One-touch System Glucose Analyzer”(Lifescan Johnson & Johnson, Milpitas, Calif., USA) after propercalibration.

Examples 1 to 8 are comparative examples involving eight rabbits.Examples 9 to 14 are examples according to the present inventioninvolving five rabbits.

In comparative examples 1 and 2 (control experiment #1 summarized inTable I) an aqueous solution of human recombinant insulin was introducedintra muscularly into two rabbits at a dose of 12 UI per animal. Incomparative examples 3 and 4 (control experiment #2 summarized in TableII), the rabbits remained intact (i.e., no insulin or other injectionwas provided to the two rabbits). In comparative examples 5 and 6(control experiment #3 summarized in Table 11), a suspension of thecrystallized dextran microparticles without insulin was provided orallyto two rabbits. In comparative examples 7 and 8 (control experiment #4summarized in Table IV), a suspension of the commercially obtainedSephadex G-150 microparticles with insulin (24 UI) was provided orallyto two rabbits. According to the Amersham Biosciences website, Sephadex®G-150 microparticles are beaded gel microparticles having a diameter of20 to 150 microns, prepared by cross linking dextran withepichlorohydrin. In examples 9-14 according to a preferred embodiment ofthe present invention (summarized in Table V), a suspension ofcrystallized dextran microparticles with insulin (24 UI) was providedorally to five rabbits. The results are summarized in Tables I-V below.

TABLE I Rabbit/ Insulin 0 min 30 min 60 min 90 min 120 min Example #dose mg/dl mg/dl mg/dl mg/dl mg/dl #1 12 UI i.m. 91 68 58 49 51 #2 12 UIi.m. 87 65 64 57 58

TABLE II Rabbit/ Insulin 0 min 30 min 60 min 90 min 120 min Example #dose mg/dl mg/dl mg/dl mg/dl mg/dl #3 0.0 98 87 87 89 86 #4 0.0 88 90 9194 87

TABLE III Rabbit/ Insulin 0 min 30 min 60 min 90 min 120 min Example #dose mg/dl mg/dl mg/dl mg/dl mg/dl #5 0.0 92 99 94 95 92 #6 0.0 90 93 9393 92

TABLE IV Rabbit/ Insulin 0 min 30 min 60 min 90 min 120 min Example #dose mg/dl mg/dl mg/dl mg/dl mg/dl #7 24 UI per os 85 82 86 81 83 #8 24UI per os 84 75 86 76 77

TABLE V Rabbit/ Insulin 0 min 30 min 60 min 90 min 120 min Example #dose mg/dl mg/dl mg/dl mg/dl mg/dl #9  24 UI per os 94 68 59 58 57 #1024 UI per os 93 64 52 54 52 #11 24 UI per os 78 52 51 49 48 #12 24 UIper os 92 64 52 53 47 #13 24 UI per os 89 53 48 38 49 #14 24 UI per os97 60 38 54 52

The data in Tables I-V show that average reduction of sugar (i.e.,glucose) in the blood of mammals is comparable when 12 UI of insulin isadministered by intramuscular injection (examples 1-2) and 24 UI ofinsulin is administered per dose (i.e., orally) with crystallizeddextran microparticles (examples 9-14). The maximum glucose reductionwas about 35 to about 60 percent at 60 min after oral administration.The concentration profile of glucose is practically the same in both theinjection and oral delivery modes. It should be noted that other amountsof insulin may be administered. For example, 30 UI of insulin may beadministered. In general, oral administration of two to three times theinsulin compared to the amount of injected insulin produces a similardrop in blood sugar for up to three hours.

It is a well known fact that insulin by itself is degraded by intestinalenzymes and is not absorbed intact across the gastrointestinal mucosa(Amidon G L, Lee H J, Absorption of peptide and peptidomimetic drugs,Ann. Rev. Pharmacol. Toxicol. 1994; 34: 321-41). However, examples 9-14show that crystallized dextran microparticles can be used as a vehiclefor oral delivery of proteins, such as insulin because the hypoglycemiaeffect received was significant. Without wishing to be bound by aparticular theory or mode of action, the present inventor believes thatthe use of the porous, crystallized dextran microparticles as an insulindelivery matrix in an aqueous suspension protected the insulin fromsignificant degradation by intestinal enzymes and allowed the insulin tobe absorbed intact across the gastrointestinal mucosa. The insulin maybe located in micropores in the microporous microparticles and/or inmacropores between the microparticles. In contrast, the use ofcross-linked Sephadex G-150 dextran microparticles with insulin (TableIV, examples 7-8) did not produce an appreciable reduction in bloodglucose concentration.

As provided in examples 9-14, the blood glucose concentration in themammal is lowered by at least 5 percent, preferably at least 30 percent,60 minutes after administering the composition containing thecrystallized dextran microparticles and insulin to the mammal (i.e., theblood glucose value in the mammal at 60 minutes after administration ofthe suspension is at least 5 percent, preferably at least 30 percentlower than that measured right before administration of the suspension).Preferably, the blood glucose concentration in the mammal is lowered byat least 5 percent, such as at least 30 percent, preferably by about 35to about 40 percent 30 minutes after administering the composition tothe mammal. Preferably, the blood glucose concentration in the mammal islowered by about 35 to about 60 percent, for example 35 to 45 percent 60minutes after administering the suspension to the mammal. Morepreferably, the blood glucose concentration in the mammal is loweredduring the entire period ranging from 30 to 240 minutes, such as 30 to120 minutes, after administering the composition to the mammal comparedto the blood glucose concentration right before administration. Forexample, the blood glucose concentration in the mammal is preferablylowered by at least 10 percent, preferably at least 30 percent, morepreferably by at least 35 percent, such as by 35 to 45 percent during aperiod ranging from 30 to 240 minutes, preferably 30 to 120 minutesafter administering the composition to the mammal.

Thus, a preferred embodiment of the present invention provides a methodof lowering blood glucose in a mammal by orally administering atherapeutically effective amount of a composition comprised ofcrystallized dextran microparticles and insulin. A “therapeuticallyeffective” amount of the compositions can be determined by prevention oramelioration of adverse conditions or symptoms of diseases, injuries ordisorders being treated. Preferably, the composition comprises anaqueous suspension of crystallized dextran microparticles having anaverage diameter of about 0.5 to about 5 microns and insulin.Furthermore, the microparticles are preferably porous microparticleswhich are crystallized prior to adding the insulin to the suspension,such that the insulin is located in contact with a surface of themicroparticles and/or in pores of the microparticles.

The crystallized microparticles preferably are comprised of dextranmolecules (i.e., polymer molecules) that are held together by aplurality of hydrogen bonds, Van Der Waals forces and/or ionic bonds andhaving substantially no covalent bonds between the dextran molecules.Thus, the molecules in the microparticles are preferably notintentionally cross-linked (i.e., a cross linking step is not carriedout) and the microparticles contain no covalent bonds between moleculesor less than 10% covalent bonds between molecules.

While a one phase composition 54 comprising insulin and microparticlesis illustrated in FIG. 10, a two phase composition, described above andillustrated in FIGS. 7, 8, 9A, 9B and 9C may also be used. For example,a two phase composition comprising a dextran phase, a PEG phase,crystallized dextran microparticles and insulin may be used. In vivo,the composition has a self assembled capsule structure comprising acrystallized dextran microparticle containing wall or shell and a PEGand insulin containing core.

Preferably, the mammal receiving the oral administration of insulincomprises a human in need of lowering blood glucose, such as a humansuffering from diabetes. Thus, the preferred embodiment of the presentinvention provides a method of treating diabetes in a human in need ofthe treatment by orally administering the suspension of insulin andcrystallized dextran microparticles described above.

Any therapeutically effective amount of insulin may be administered tothe mammal. The amount of insulin may vary based on the type of mammal(i.e., human or rabbit), the weight of the mammal, the composition ofthe suspension, the amount of desired reduction of blood glucose andother factors. One non-limiting example of insulin content in thesuspension is about 10 UI to about 2,500 UI of human recombinant insulinper one gram of suspension, such as about 12 UI to about 30 UI, such as24 UI of human recombinant insulin. However, this amount may vary asdesired.

The present invention should not be considered limited to the preferredembodiments described above. Other matrix material may be used for oralinsulin delivery, such as organic or inorganic microporous particles.Preferably, the particles are microparticles which enhance insulinpenetration through gastrointestinal mucosa and/or which stabilize thecomposition. Furthermore, while the suspension preferably contains onlywater solvent; a matrix; and an insulin solution or suspension; thedelivery system may also contain additional materials. For example, thecomposition may contain a second phase such as the PEG phase of a twophase system. Thus, another preferred aspect of the present inventionincludes a method of lowering blood glucose in a mammal comprising oforally administering a composition comprising a therapeuticallyeffective amount of insulin and a matrix material to the mammal to lowerblood glucose of the mammal by at least 30 percent 60 minutes afteradministering the suspension to the mammal. In another preferred aspectof the present invention, a method of administering a suspension to amammal comprised of orally administering an aqueous suspension ofcrystallized dextran microparticles and a therapeutically effectiveamount of insulin to the mammal.

As noted above, the crystallized dextran microparticles used as a matrixmaterial for oral administration of insulin or other protein based drugsmay be made by any suitable method (see FIG. 1, for example).Preferably, the microparticles are made by the process of any of thepreferred embodiments described herein. Preferably, but not necessarily,the microparticles are formed in an aqueous solution without using anorganic solvent. Thus, in a preferred aspect of the present invention, atherapeutically effective amount of insulin and the crystallized dextranmicroparticles are combined in water after the microparticles have beencrystallized to form an aqueous suspension of insulin and crystallizeddextran microparticles. The microparticles may be added to the waterbefore, at the same time and/or after adding the insulin to the water.Furthermore, the microparticles may be orally administered to a mammalin the solvent in which they were formed. Alternatively, they may beremoved from the solvent in which they were formed and placed into wateror other aqueous solutions for oral administration or dried and providedin solid form, such as tablet or capsule, for oral administration.

The aqueous suspension of crystallized dextran microparticles and atherapeutically effective amount of insulin (or other suitablesuspensions of insulin and a matrix material, such as a suspension ofinsulin and microporous microparticles) is preferably provided as adosed pharmaceutical composition which is dosed for oral administrationto a human. In one preferred aspect of the present invention, thecomposition is located in a vessel in an amount dosed for a single oraladministration to a human. The vessel may comprise any container whichmay hold a suspension, such as a plastic or glass bottle, a tube, adropper, a spray nozzle, a pouch and/or other suitable vessels. Thisvessel contains an amount of suspension sufficient for a single oraldose of the composition.

In another preferred aspect of the present invention, the composition islocated in any suitable vessel in an amount suitable for multiple oraladministration doses. The vessel contains an instruction for oral dosageadministration to a human. The instruction may be printed on the vessel,such being as printed directly on the vessel or on a label attached tothe vessel, or enclosed with the vessel, such being printed on a sheetof paper enclosed with the vessel in a cardboard box or in a pharmacyenvelope. The instructions may describe the amount of the compositionthat should be taken with each dose, the frequency that the dose shouldbe taken, how to measure the dose of the composition for oraladministration and/or any other suitable oral drug instructions for anadministering health care practitioner and/or a patient in need of thedrug. Alternatively, the instructions may comprise directions forelectronically or audibly accessing the dosing and administrationinstructions, such as a link to a website containing the instructions ora telephone number or recording where the instructions are providedaudibly.

In another preferred aspect of the present invention, the aqueoussuspension of crystallized dextran microparticles and a therapeuticallyeffective amount of insulin located in a vessel are provided in apharmaceutical composition kit with instructions for oral administrationof the composition to a human in need thereof. The kit may compriseinstructions printed on the vessel or on a label attached to the vesselor a sheet of paper enclosed with the vessel, such as in a cardboard boxor pharmacy envelope including a bottle (i.e., vessel) and the sheet ofinstructions.

It should be noted that the composition for oral administration may bein the form of an aqueous suspension, but other delivery forms may beused to lower the blood glucose in a mammal. For example, the porouscrystallized dextran microparticles and the insulin may be orallyadministered in the form of a tablet or a capsule.

To orally administer the composition in solid form to a mammal, such asa human, the solution of crystallized dextran microparticles and insulinis first dried, such as freeze dried, to form a powder. The powder maythen be compressed into a tablet, along with optional pharmaceuticallyacceptable excipients or placed into a pharmaceutically acceptablecapsule.

D. Materials

In the preferred embodiments of the present invention, the therapeuticagent comprises insulin. In other words, the therapeutic agent mayconsist essentially of insulin alone or comprise insulin in combinationwith another agent. The term “insulin” shall be interpreted to encompassinsulin analogs, natural extracted human insulin, recombinant producedhuman insulin, insulin extracted from bovine and/or porcine sources,recombinant produced porcine and bovine insulin and mixtures of any ofthese insulin products. The term is intended to encompass thepolypeptide normally used in the treatment of diabetics in asubstantially purified form but encompasses the use of the term in itscommercially available pharmaceutical form, which includes additionalexcipients. The insulin is preferably recombinant produced and may bedehydrated (completely dried) or in solution.

The terms “insulin analog,” “monomeric insulin” and the like are usedinterchangeably herein and are intended to encompass any form of“insulin” as defined above, wherein one or more of the amino acidswithin the polypeptide chain has been replaced with an alternative aminoacid and/or wherein one or more of the amino acids has been deleted orwherein one or more additional amino acids has been added to thepolypeptide chain or amino acid sequences, which act as insulin indecreasing blood glucose levels. In general, the term “insulin analogs”of the preferred embodiments of the present invention include “insulinlispro analogs,” as disclosed in U.S. Pat. No. 5,547,929, incorporatedhereinto by reference in its entirety; insulin analogs including LysProinsulin and humalog insulin, and other “super insulin analogs”, whereinthe ability of the insulin analog to affect serum glucose levels issubstantially enhanced as compared with conventional insulin as well ashepatoselective insulin analogs which are more active in the liver thanin adipose tissue. Preferred analogs are monomeric insulin analogs,which are insulin-like compounds used for the same general purpose asinsulin, such as insulin lispro, i.e., compounds which are administeredto reduce blood glucose levels.

The term “analog” refers to a molecule, which shares a common functionalactivity with the molecule to which it is deemed to be comparable andtypically shares common structural features as well.

The term “recombinant” refers to any type of cloned therapeuticexpressed in prokaryotic cells or a genetically engineered molecule, orcombinatorial library of molecules which may be further processed intoanother state to form a second combinatorial library, especiallymolecules that contain protecting groups which enhance thephysicochemical, pharmacological, and clinical safety of the therapeuticagent.

The term dextran microparticles includes unsubstituted dextranmicroparticles and substituted dextran microparticles. For example,substituted dextran microparticles include dextran substituted with asuitable group, such as a methyl group, up to a degree which does nothamper crystallization of the dextran microparticles, such as up to 3.5or less percent branching. The average microparticle diameter ispreferably about 0.5 to about 5 microns, more preferably about 1 toabout 2 microns.

Furthermore, while porous non cross-linked dextran microparticles, suchas crystallized microparticles, are preferably used with the therapeuticagent, other suitable organic or inorganic microparticles may be usedinstead, such as other polymer microparticles including polysaccharides,PLA, PLGA, PMMA, polyimides, polyesters, acrylates, acrylamides, vinylacetate or other polymeric materials, biomaterial particles such asalginate and cells, or inorganic particles, such as silica, glass orcalcium phosphates. Preferably the microparticles are biodegradable.Preferably, porous microparticles are used. Most preferably, themicroparticles have sufficient porosity to contain the therapeutic agentwithin the pores and to provide a timed release of the therapeutic agentfrom the pores. In other words, the therapeutic agent is released overtime from the pores, such as in over 5 minutes, preferably in over 30minutes, most preferably in over one hour, such as in several hours toseveral days, rather than all at once. Thus, the particle material, poresize and pore volume can be selected based on the type of therapeuticagent used, the volume of therapeutic agent needed for delivery, theduration of the delivery of the therapeutic agent, the environment wherethe therapeutic agent will be delivered and other factors.

Thus, in a preferred aspect of the present invention, the therapeuticagent is located at least partially in the pores of the porousmicroparticles. Preferably, the therapeutic agent is not encapsulated inthe microparticle (i.e., the microparticle does not act as a shell witha therapeutic agent core inside the shell) and is not attached to thesurface of the microparticle. However, if desired, a portion of thetherapeutic agent may also be encapsulated in a microparticle shelland/or is attached to the surface of the microparticle in addition tobeing located in the pores of the microparticle. The location of thetherapeutic agent in the pores provides an optimum timed release of thetherapeutic agent. In contrast, the therapeutic agent attached to thesurface of the microparticle is often released too quickly, while thetherapeutic agent encapsulated in the microparticle is often notreleased soon enough and is then released all at once as themicroparticle shell disintegrates. In a two phase system, at least 80%of the therapeutic agent is preferably located in a core surrounded by awall or shell comprising the microparticles.

E. Methods of Making

The microparticles may be formed by any suitable method. Preferably, themicroparticles are combined with the therapeutic agent after themicroparticles are formed. Thus, the microparticles, such as thecrystallized dextran microparticles are formed by any suitable methodand then the therapeutic agent and the microparticles are combined byany suitable method. In contrast, in some prior art methods, thetherapeutic agent is encapsulated into a microparticle shell byproviding the particle precursor material and the therapeutic agent intoa solution and then crystallizing or cross-linking the precursormaterial, such as a monomer or oligomer material, to encapsulate atherapeutic agent core into a microparticle shell.

Preferably, the therapeutic agent is provided into the pores of theporous microparticles after the microparticles are formed. Thus, theporous microparticles are first formed and then the therapeutic agent isprovided into a solution containing the microparticles to allow thetherapeutic agent to permeate into the pores of the microparticles. Ofcourse, some of the therapeutic agent may also become attached to thesurface of the microparticle in this process.

Thus, a method to manufacture non cross-linked, porous crystallizeddextran microparticles includes preparation of a dextran solution, suchas an aqueous dextran solution, conducting a crystallization process toform crystallized porous dextran microparticles, and if desired,isolating crystallized porous dextran microparticles from the solution.A therapeutic agent is then permeated into the pores of themicroparticles by providing the therapeutic agent into thecrystallization solution containing the microparticles or by providingthe isolated microparticles and the therapeutic agent into a secondsolution, such as a second aqueous solution. For example, crystallizeddextran microparticles may be formed in a first, low molecular weightdextran aqueous solution, such as a 2 to 20 kDa dextran solution. Themicroparticles are then removed from the first solution and then placedinto a second dextran aqueous solution having a higher molecular weightdextran, such as a 40 to 500 kDa solution, for example, a 40 to 75 kDasolution. The second solution may comprise a first phase of a two phasesystem, which is then combined with a second phase, such as a PEG phasecontaining a therapeutic agent. A similar method may be used with otherporous microparticles, where a therapeutic agent is then permeated intothe pores of the microparticles after the porous microparticles areformed by any suitable microparticle formation method, including, butnot limited to crystallization. The components of the composition suchas insulin, microparticles and one or more aqueous phases may becombined in any suitable order sequentially or simultaneously.

Preferably, the microparticles are formed by self assembly from asolution that does not contain organic solvents and organic reactionpromoters which leave an organic residue in the microparticles. Thus,for example, the dextran microparticles are preferably formed by selfassembly from an aqueous dextran solution. However, if desired, organicsolvents and/or organic reaction promoters may also be used. In thiscase, the microparticles may be purified prior to subsequent use toremove the harmful organic residue.

As described above, the capsule structure having a first phase core anda second phase wall or shell may be formed in vivo or in vitro from atwo phase composition. The composition may be a dried powder, such asfreeze dried and stored as a powder or porous cake. When the compositionis ready to be administered to a mammal, it is hydrated and administeredto a mammal orally.

Preferably, the composition which includes the microparticles and thetherapeutic agent is a flowable colloidal system. Examples of flowablecolloidal systems include emulsions and suspensions. In contrast, someprior art compositions include a therapeutic agent in a dextran hydrogelor in a cross-linked dextran matrix. A dextran hydrogel and across-linked dextran matrix are not flowable compositions if notspecifically prepared.

In another preferred aspect of the present invention, the microparticlescomprise microparticles which are adhesive to mammalian mucosa.Preferably the adhesive microparticles are porous microparticlesdescribed above. This further improves the effective delivery of thetherapeutic agent.

In another preferred aspect of the present invention, the microparticlescomprise microparticles whose surface has been specially modified toenhance the adhesion of the therapeutic agent to the microparticlesurface and to optimize the delivery of the therapeutic agent. Themicroparticle surface may contain any suitable modification that wouldincrease the adhesion of the therapeutic agent.

The foregoing description of the invention has been presented forpurposes of illustration and description. It is not intended to beexhaustive or to limit the invention to the precise form disclosed, andmodifications and variations are possible in light of the aboveteachings or may be acquired from practice of the invention. Thedrawings and description were chosen in order to explain the principlesof the invention and its practical application. It is intended that thescope of the invention be defined by the claims appended hereto, andtheir equivalents.

All of the publications and patent applications and patents cited inthis specification are herein incorporated in their entirety byreference.

1. An implant produced by providing a liquid composition comprising anaqueous solution of dextran with a molecular weight of 1.0 to 200 kDa ata concentration of 40-65% by weight to a mammal by introducing theliquid composition into the body of the mammal, thereby forming animplant in the body of the mammal.
 2. The implant of claim 1, whereinthe liquid composition further comprises a therapeutic agent.
 3. Theimplant of claim 2, wherein the therapeutic agent is present at aconcentration of 15-30% by weight.
 4. The implant of claim 2, whereinthe therapeutic agent is a protein.
 5. The implant of claim 1 or 2,wherein the dextran is present at a concentration from 55-65% by weight.6. The implant of claim 1 or 2, wherein the liquid composition isintroduced into the body of the mammal by injection.
 7. The implant ofclaim 1 or 2, wherein said liquid composition comprises a rehydratedfreeze-dried dextran component.
 8. The implant of claim 2, wherein thetherapeutic agent is insulin.
 9. A method of treating a disease ordisorder in a mammal, comprising forming the implant of claim 2 in thebody of the mammal, wherein said implant releases the therapeutic agentinto the body of the mammal.
 10. The method of claim 9, wherein thedisease or disorder is diabetes.
 11. The method of claim 9, wherein thetherapeutic agent is insulin.
 12. The method of claim 9, wherein themammal is a human.